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A comparison of 2‐D chromatography separations using UV and 18 O quantification of proteins in similar proteomes
Author(s) -
Smith Julia R.,
Matus Isaac,
Greene Andrew S.
Publication year - 2008
Publication title -
journal of separation science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.72
H-Index - 102
eISSN - 1615-9314
pISSN - 1615-9306
DOI - 10.1002/jssc.200700293
Subject(s) - chromatography , chemistry , fractionation , proteome , trypsinization , peptide , mass spectrometry , analytical chemistry (journal) , trypsin , biochemistry , enzyme
Proteins present in two mitochondria preparations were separated by 2‐D chromatography using the ProteomeLab TM PF‐2D Protein Fractional System, protein fractionation in two dimensions (PF‐2D). The proteins in each first‐dimension fraction were determined by trypsinization and LC‐MS/MS. Chromatography peaks were quantified by UV detection using the “Mapping Tools” software (Beckman). The proteins present in UV detected peaks were trypsinized and identified by automated MS/MS sequencing. Relative amounts of the proteins present in the equivalent peak for each sample were assessed by comparison of the intensities of the constituent peptides and a predicted PF‐2D value was calculated from the total ion count (TIC) for each peptide. Relative quantification for 18 O labeled peptides was performed using the ZoomQuant (v1.43b) software [1, 2]. We found that the chromatography peaks detected by UV generally contained several proteins. Using 18 O labeling we determined that in each peak the ratios of the constituent proteins were different. When these ratios were normalized using the TIC to account for abundance, the resulting ratio corresponded to that determined by UV. The predicted value for the PF‐2D score corresponded to the observed value for each peak irrespective of the number or proteins detected.